Sample Preparation for Chromium Linked-reads
For the genomics application, high quality HMW DNA is required. DNA will be tested on the Fragment Analyzer using the 50Kb protocol and, if needed, on the PippinPuls (a PFGE system).
10X Genomics recommendations for specific HMW DNA isolation protocols can be accessed from here.
- DNA should be above 50kb with greater than 80% of the DNA at 100kb or larger. After isolation, the DNA should be handled very gently (minimal pipetting, use of wide-bore tips, avoid freeze/thaw, etc).
- Ship HMW DNA on dry ice for stability, but once it arrives at the GSL it will be stored at 4C.
- Though the protocol uses 1ng of HMW DNA as input, a minimum of 500ng of DNA in 20ul of water or Tris should be submitted to allow for QC and visualization of the HMW DNA on a gel.
- If you are are submitting 24 or more samples please submit samples in a 96-well plate and include an Excel file with the sample layout in your order.
- PLEASE USE V BOTTOM PLATES AND NOT ROUND/FLAT BOTTOM PLATES.
Sample Preparation for Chromium Single Cell and nuclei RNAseq
- For pricing please see our website at the link here for 10X library prep and the link here for NextSeq runs. Official quotes can be provided upon request.
- We can process up to 8 samples on a 10X chip at a time. The library prep cost is reflective of that and cost per sample is lowest for 8 samples. We follow 10X’s sequencing recommendations, which you can view at the link here. Read lengths can be changed as desired, but please note that shorter transcript reads can reduce alignment rates. Generally a minimum of 20,000 read pairs per cell is needed. The type and number of sequencing run(s) depends on the number of samples and coverage you need. We can make libraries with between 500 and 10,000 cells per sample, though the recommended starting point is 1,600 cells the first time you do the experiment. It is recommended to try the protocol with just one sample first before moving forward with a larger number.
- 10X has a list of demonstrated cell prep protocols at the link here. Customers should work with the tissue in their lab and provide us with a prepared cell suspension. Cell preparation methods can vary between different tissue types. Please refer to 10X’s recommendations and check the literature to find methods appropriate for your sample type. Cells are preferable in most cases, but nuclei can be used as well. Feel free to reach out to 10X or GGBC for further recommendations.
- When you submit your order and have the cell suspension ready for library prep we need an accurate measure of cell concentration and viability. These QC data are critical to the protocol working correctly. The ideal concentration is between 700-1,200 cells/uL and the viability should be >90%. An inaccurate concentration could clog the chip or result in low yield, and the presence of dead cells will reduce the recovery rate and result in a high level of noise in the data. It is also important to minimize the time from cell prep to loading the 10X chip.
- We do not have the equipment for cell counting and viability here at GGBC, so we have to rely on your data. We’ve seen the best results from customers who prep their cells, sort and count by flow cytometry, and then bring them on ice immediately to our lab. There is a flow cytometer in another core lab on campus that you can use. We’ve also had good results from a few customers who did the final steps of cell prep in our lab before counting and assessing viability by microscopy immediately prior to loading the 10X chip. If you want to go that route you would need to bring a scope and whatever other materials you need.
- Please contact Julia Portocarrero at least 2 weeks in advance before submitting a single cell order.